 What is genetic material? Griffith experiment 1928

DNA  Watson-Crick model 1953

 DNA polymerase I and III  DNA ligases  Primase  DNA replication is semiconservative!

Meselson-Stahl experiment 1958

oriC and dnaA Boxes

 Cis acting sites: on this side of (acting only on the DNA, they made of  Trans acting (proteins) : on the other side of (acting on any DNA)  dnaA, B, C, G,

Termination

 Bacterial genetic information: On bacterial DNA On bacterial DNA On plasmids On plasmids On bacteriphages On bacteriphages On transposons On transposons

BActerial Genome -Usually 1 chromosome Circular or linear No histon proteins

 In circular bacterial DNA the replication begins at the ori locus  Ends at ter locus

 Plasmids: ds DNA; circular Various copy number bp long Carry genes providing advantages for the bacterium

 Transposons (IS seequences) Can couple their replication to the cell division - Their propagation depends on the integration with the bacterial replicon - The insertion sites are not spesific

 Bacteriophages Viruses of the bacteria Ds/ss DNA,ds/ss RNA Lytic or temperate phages (prophage) Different propagation strategies

Gene transfer among bacteria  Vertical transfer  Lateral or horizontal transfer - conjugation - transduction - transformation

Conjugation Most frequently plasmids are transferred Tra gene products are needed F+ E. Coli Sex pilus Hfr R plasmids

Interrupted Mating  Chromosome transfer from the Hfr into the F- is slow: it takes about 100 minutes to transfer the entire chromosome.  The conjugation process can be interrupted using a kitchen blender.  By interrupting the mating at various times you can determine the proportion of F- cells that have received a given marker.  This technique can be used to make a map of the circular E. coli chromosome.

Transduction  General Phage Life Cycle 1. Phage attaches to the cell and injects its DNA.  2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins.  3. New phage particles are assembled.  4. Cell is lysed, releasing about 200 new phage particles.  Total time = about 15 minutes.

Generalized Transduction  Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA.  These chromosomal pieces are quite small: about 1 1/2 minutes of the E. coli chromosome, which has a total length of 100 minutes.  A phage containing E. coli DNA can infect a fresh host, because the binding to the cell surface and injection of DNA is caused by the phage proteins.  After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome).  A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated.

Transduction Mapping  Only a small amount of chromosome, a few genes, can be transferred by transduction. The closer 2 genes are to each other, the more likely they are to be transduced by the same phage. Thus, “co-transduction frequency” is the key parameter used in mapping genes by transduction.  Transduction mapping is for fine-scale mapping only. Conjugation mapping is used for mapping the major features of the entire chromosome.

Specialized Transduction  Some phages can transfer only particular genes to other bacteria.  Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle.  lambda has 2 distinct phases of its life cycle. The “lytic” phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage.

Lysogenic Phase  The “lysogenic” phase of the lambda life cycle starts the same way: the lambda phage binds to the bacterial cell and injects its DNA. Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr.  Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the “prophage”.  After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs.  When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell.

Transduction  Phage mediated recombination

Transformation Competent cells Artificially (forced) with CaCl or temperature shock

Genetıc Mapping  Sequencing of the gene  Cloning the gene  Gene labeled  Hybridization  Localization of the gene on the bacterial genome

 Nucleic acid amplification - PCR and other technologies - Real time PCR